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Increasingly complex metabolic pathways have been engineered by modifying natural pathways and establishing de novo pathways with enzymes from a variety of organisms. Here we apply retro-biosynthetic screening to a modular pathway design to identify a redox neutral, theoretically high yielding route to a branched C6 alcohol. Enzymes capable of converting natural E. coli metabolites into 4-methyl-pentanol (4MP) via coenzyme A (CoA)-dependent chemistry were taken from nine different organisms to form a ten-step de novo pathway. Selectivity for 4MP is enhanced through the use of key enzymes acting on acyl-CoA intermediates, a carboxylic acid reductase from Nocardia iowensis and an alcohol dehydrogenase from Leifsonia sp. strain S749. One implementation of the full pathway from glucose demonstrates selective carbon chain extension and acid reduction with 4MP constituting 81% (90±7 mg l⁻¹) of the observed alcohol products. The highest observed 4MP titre is 192±23 mg l⁻¹. These results demonstrate the ability of modular pathway screening to facilitate de novo pathway engineering.United States. Army Research Office (W911NF-09-0001

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol